DNA was extracted and purified from skin swabs as described previously (Schowalter et al., 2010 (link)). The DNA was amplified by multiply primed rolling circle amplification (RCA) using the Illustra TempliPhi 100 Amplification Kit according to the manufacturer’s recommendations (GE Healthcare, Piscataway, NJ), with supplementation of 450 μM dNTPs as described by Rector et al. (2004) (link).
Illustra templiphi 100 amplification kit
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Illustra TempliPhi 100 Amplification Kit is a product designed for rapid and accurate amplification of DNA samples. It utilizes a proprietary phi29 DNA polymerase-based technology to achieve isothermal whole genome amplification, generating microgram quantities of DNA from nanogram-level starting material. The kit is suitable for a wide range of applications, including next-generation sequencing, genotyping, and other molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Milli q purification system, by Merck Group (4 mentions)
Mach1, by Thermo Fisher Scientific (4 mentions)
Plasmid safe atp dependent dnase, by Illumina (3 mentions)
Nuclease free water, by Qiagen (3 mentions)
Gblock gene fragment, by Integrated DNA Technologies (2 mentions)
Phusion u hot start dna polymerase, by Thermo Fisher Scientific (2 mentions)
Hyclone water, by GE Healthcare (2 mentions)
Phusion u hot start, by Thermo Fisher Scientific (2 mentions)
Phusion hot start 2 dna polymerase, by Thermo Fisher Scientific (2 mentions)
Easypure plant genomic dna kit, by Transgene (1 mentions)
Magna pure compact nucleic acid isolation kit, by Roche (1 mentions)
Pcr clean up gel extraction kit, by Macherey-Nagel (1 mentions)
Platinum taq dna polymerase high fidelity kit, by Thermo Fisher Scientific (1 mentions)
Plasmid plus midi kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
19 protocols using illustra templiphi 100 amplification kit
1
DNA Amplification by Rolling Circle Amplification
2
Amplification and Profiling of Extrachromosomal DNA
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Extrachromosomal circular DNA (eccDNA) was isolated from 3 µg of genomic DNA and amplified according to the previously described protocol [40 (link)], with several modifications [8 (link)]. For double-stranded linear DNA digestion, genomic DNA was treated with Plasmid-Safe ATP-Dependent DNAse (Epicenter, Madison, WI, USA) for 120 h at 37 °C, with an extra amount of reagents added (10 units of Plasmid-Safe ATP-Dependent DNAse; 2 µL 25 mM ATP; 0.3 µL Plasmid-Safe 10x Reaction Buffer) after 48 and 96 h. DNA precipitation was carried out by adding 0.1 volume 3 M sodium acetate (pH = 5.2) and 2.5 volume absolute ethanol, followed by overnight incubation at −20 °C. Precipitated eccDNA was amplified by random rolling circle amplification (RCA) reaction using the Illustra TempliPhi 100 Amplification Kit (GE Healthcare, Chicago, IL, USA) for 65 h at 28 °C. RCA reaction products were diluted five times and exposed to inverse PCR with the RTE-specific primers listed in Table 1 .
The PCR conditions were: 94 °C for 1 min; 35 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min; and a final elongation at 72 °C for 3 min.
The PCR conditions were: 94 °C for 1 min; 35 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min; and a final elongation at 72 °C for 3 min.
3
DNA Extraction and RCA Analysis
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Total DNA was extracted from each of the AGV-positive samples using Easy Pure Plant Genomic DNA Kit (TransGen, Beijing, China) following the manufacturer’s instructions. The extracted DNA was used as template in RCA, which was performed using Illustra TempliPhi 100 Amplification kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions. The RCA products were digested with the restriction enzyme BamHI and separated on 1% agarose by electrophoresis.
4
DNA Amplification and Digestion Protocol
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DNA was amplified by multiple-primed rolling-circle amplification (RCA) using the Illustra TempliPhi 100 amplification kit (GE Healthcare, Little Chalfont, UK) in agreement with the protocol indicated by Rector and colleagues [48 (link)]. Then, restriction enzymes BamHI and HindIII were used for performing digestion. The obtained products were run on a 0.8% agarose gel to confirm the presence of DNA fragments consistent with the length of a papillomaviral genome.
5
DNA Extraction and Genome Enrichment
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DNA was extracted using the MagNA Pure Compact Nucleic Acid Isolation kit (Roche, USA) and circular genomes enriched using the Illustra TempliPhi 100 Amplification kit (GE Healthcare, Amersham, UK), according to the manufacturer's instructions.
6
Circular DNA Amplification and Cloning
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RCA method, based on the Phi29 DNA polymerase from Illustra™ TempliPhi 100 Amplification kit (GE Life Sciences, USA),
was used to amplify the circular DNA molecules of WDV genome from total DNAs of WDV- infected samples as described by Haible et al. ( 33 (link)
). RCA products were then digested using HindIII and EcoRI enzymes (Thermo Scientific, Germany) which were expected
to be single-cutters according to the results of in silico analysis of Iranian WDV sequences using NEBcutter software
(ver. 2) (http://nc2.neb.com/ NEBcutter2/ ).
The released DNA fragments with a size equal to or lower than ~2.7 Kb were
recovered from agarose gel using the PCR clean-up Gel extraction kit (Macherey-Nagel, Germany).
These fragments were then cloned into a HindIII- or EcoRI-linearized pBluescript II KS+ plasmid (Stratagene,
USA) using the standard cloning protocol ( 37
). Bacterial colonies harboring the recombinant plasmid were detected by PCR using a WDV-specific primer pair (Table 2 ).
The inserts were sequenced on both strands by sequencing service of BIONEER (South Korea) using universal M13/M13-Reverse primer pair given inTable 2 .
was used to amplify the circular DNA molecules of WDV genome from total DNAs of WDV- infected samples as described by Haible et al. ( 33 (link)
). RCA products were then digested using HindIII and EcoRI enzymes (Thermo Scientific, Germany) which were expected
to be single-cutters according to the results of in silico analysis of Iranian WDV sequences using NEBcutter software
(ver. 2) (
The released DNA fragments with a size equal to or lower than ~2.7 Kb were
recovered from agarose gel using the PCR clean-up Gel extraction kit (Macherey-Nagel, Germany).
These fragments were then cloned into a HindIII- or EcoRI-linearized pBluescript II KS+ plasmid (Stratagene,
USA) using the standard cloning protocol ( 37
). Bacterial colonies harboring the recombinant plasmid were detected by PCR using a WDV-specific primer pair (
The inserts were sequenced on both strands by sequencing service of BIONEER (South Korea) using universal M13/M13-Reverse primer pair given in
7
HPV DNA Amplification via RCA
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DNA was extracted and purified from a HPV-ICB2-positive skin swab as described previously (Schowalter et al., 2010 (link)). The DNA was amplified by multiply-primed rolling circle amplification (RCA) with random hexamer primers using an Illustra TempliPhi 100 Amplification kit following the manufacturer’s recommendations (GE Healthcare, Piscataway, NJ), with supplementation of 450 μM dNTPs as described by Rector et al. (2004) (link).
8
Amplifying PsuPV1 L1 Gene Genomes
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Three randomly selected PsuPV1 L1 gene–positive samples were first subjected to rolling circle amplification (RCA) using an Illustra TempliPhi 100 Amplification Kit (GE Healthcare Life Sciences, Little Chalfont, UK), following the manufacturer’s instructions. The PsuPV1 type-specific primer set (PsuPV1_LNGL1-F: 5′-CGTTGCTAAACAAACTAGGTGAC-3′ and PsuPV1_LNGL1-R: 5′-GATGTCCGGTTGTCCCTAC-3′), targeting the PsuPV1 L1 gene, was subsequently used to amplify their complete viral genomes, with the help of inverted long-range PCR in combination with a Platinum Taq DNA Polymerase High Fidelity Kit (Invitrogen, Carlsbad, CA, USA), as described previously [31 (link)].
Sanger sequencing of the PCR products obtained was performed by a primer-walking strategy, using 22 sequencing primers, as already described [31 (link)]. Sequences were constructed using the Vector NTI Advance v11.5.4 (Thermo Fisher Scientific, Waltham, MA, USA) and BioEdit Sequence Alignment Editor v7.2.6.1 (Ibis Therapeutics, Carlsbad, CA, USA) [35 ] software packages and compared with the PsuPV1 reference sequence (GenBank acc. no. HG939559), using the BLAST algorithm [33 (link)].
Sanger sequencing of the PCR products obtained was performed by a primer-walking strategy, using 22 sequencing primers, as already described [31 (link)]. Sequences were constructed using the Vector NTI Advance v11.5.4 (Thermo Fisher Scientific, Waltham, MA, USA) and BioEdit Sequence Alignment Editor v7.2.6.1 (Ibis Therapeutics, Carlsbad, CA, USA) [35 ] software packages and compared with the PsuPV1 reference sequence (GenBank acc. no. HG939559), using the BLAST algorithm [33 (link)].
9
Antibiotics usage and DNA manipulation protocols
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Antibiotics (Gold Biotechnology) were used at the following working concentrations: carbenicillin 50 μg/mL, spectinomycin 50 μg/mL, chloramphenicol 25 μg/mL, kanamycin 50 μg/mL, tetracycline 10 μg/mL, streptomycin 50 μg/mL. HyClone water (GE Healthcare Life Sciences) was used for PCR reactions and cloning. For all other experiments, water was purified using a MilliQ purification system (Millipore). Phusion U Hot Start DNA polymerase (Thermo Fisher Scientific) was used for all PCRs. Plasmids and SPs were cloned by USER assembly21 (link). Genes were obtained as synthesized gBlock gene fragments from Integrated DNA Technologies or PCR amplified directly from E. coli genomic DNA. Plasmids were cloned and amplified using either Mach1 (Thermo Fisher Scientific) or Turbo (New England BioLabs) cells. Unless otherwise noted, plasmid or SP DNA was amplified using the Illustra Templiphi 100 Amplification Kit (GE Healthcare Life Sciences) prior to Sanger sequencing. A full list of plasmids used in this work is given in Supplementary Table 5 Supplementary Table 6
10
Antibiotic and Nuclease-free Reagents for Molecular Biology
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